Larvicidal potential, antimicrobial properties and molecular docking analysis of Egyptian Mint (Mentha rotundifolia) against Culex pipiens L. (Diptera: Culicidae) and Midgut-borne Staphylococcus aureus

Mosquitoes prefer stagnant areas near hospitals to live and easily spread pathogenic bacteria. Our current study aims to isolate multidrug-resistant (MDR) Staphylococcus aureus isolates from midguts of Mosquito Culex pipiens and study the potential of mint as a biocontrol strategy against C. pipiens larvae and their midgut-borne S. aureus. Samples of the third and fourth larval instars of C. pipiens were collected from water ponds around three Cairo hospitals. Ciprofloxacin, gentamycin and tetracycline, as well as various concentrations of mint leaf extract (MLE) were tested for antibiotic susceptibility. Sixty-five isolates were obtained and showed antibiotic resistance to tetracycline, gentamycin, ciprofloxacin, and undiluted MLE with resistant percentages (%) of 27.69, 30.76, 17.46, and 23.08%, respectively. Undiluted MLE inhibited 61.53% of the multidrug S. aureus isolates, whereas it couldn't inhibit any of these isolates at dilutions less than 50 μg/mL. The MIC of MLE was ≤ 700 µg/mL, while the MIC of the antibiotics ranged from 0.25 to 5.0 µg/mL for the three antibiotics. The most inhibited S. aureus isolate was identified by 16SrRNA sequencing approach and registered in GenBank as S. aureus MICBURN with gene accession number OQ766965. MLE killed all larval stages after 72 h of exposure, with mortality (%) reaching 93.33 and 100% causing external hair loss, breakage of the outer cuticle epithelial layer of the abdomen, and larvae shrinkage. Histopathology of treated larvae showed destruction of all midgut cells and organelles. Gas chromatography (GC) of MLE revealed that menthol extract (35.92%) was the largest active ingredient, followed by menthone (19.85%), D-Carvone (15.46%), Pulegone (5.0579%). Docking analysis confirmed that alpha guanine and cadinol had the highest binding affinity to both predicted active sites of Culex pipiens acetylcholinesterase. As a result, alpha-guanine and cadinol might have a role as acetylcholinesterase inhibitors.


Isolation of mid-gut-borne S. aureus in C. pipiens larvae
To isolate S. aureus, 3rd and 4th instar mosquito larvae were surface sterilized by soaking in 70% ethyl alcohol for 30 s, followed by 5 washes in sterile Milli-Q water to remove excess ethyl alcohol.Under aseptic conditions, the heads and abdomens of the larvae were removed using a sterile scalpel and forceps.The extracted midguts were placed in 100 mL phosphate-buffered saline (PBS).The midguts were homogenized in 100 mL PBS using a sterile plastic pestle.The homogenates were inoculated onto Parker agar plates supplemented with 0.01% (w/v) potassium tellurite to select for S. aureus.Plates were incubated at 37 °C for 24-48 h.Rough, black colonies displaying yellow zones of precipitation, showing mannitol fermentation, were presumptively identified as S. aureus.Isolates were subcultured on Tryptic soy agar to get pure cultures for downstream testing.Antibiotic susceptibility testing was performed on the isolates using commercially available ciprofloxacin, gentamicin, and tetracycline antibiotic discs and undiluted MLE extract 15 .

Mint leaf extraction
The leaves of mint (Mentha rotundifolia) (Family: Lamiaceae) were purchased from a local market in Cairo, Egypt.The leaves were washed twice with tap water, followed by a rinse with distilled water to remove minerals and chlorine.The leaves were then dried and ground in an electrical grinding machine (Molineux, AR6801EG).For storage, the harvested mint powder was placed in airtight jars.For extraction, mint powder (10 g/100 mL) was added to isopropanol, followed by a filtration process after 6 h.To avoid contamination, the collected filtrate was evaporated in a water bath at 60 °C.The extract was filter-sterilized after evaporation of the isopropanol.

Antibiotic susceptibility test for MLE and the three commercial antibiotics against S. aureus isolates
Antibiotic susceptibility testing was performed using the disc diffusion method following CLSI guidelines.Commercially antibiotic discs containing ciprofloxacin (5 μg/disc, gentamicin (10 μg/disc), and tetracycline (25 μg/ disc) were utilized at the standard dose concentrations for antibiotic susceptibility testing, as they are commonly used to treat S. aureus infections.A control S. aureus ATCC 29737 strain was used as a positive control.Isolates and controls were inoculated onto Mueller-Hinton agar plates and incubated at 37 °C for 24 h.Inhibition zone diameters (IZD) in cm were measured and interpreted using CLSI breakpoints to determine susceptibility.Experiments were done in triplicate.The most resistant isolates were selected for further characterization.Multidrug resistance was defined as resistance to at least two antibiotics.The isolate with the highest resistance was chosen for additional studies.This approach identified antibiotic-resistant S. aureus isolates from the mosquito larvae samples 15 .The identity of the selected S. aureus isolate was definitively confirmed through molecular identification, as described in the subsequent section.

Minimum Inhibitory Concentration (MIC) of the commercial antibiotics
All S. aureus isolates were subjected to an antimicrobial susceptibility test using ciprofloxacin (5µg/mL), gentamicin (10µg/mL), and tetracycline (25 µg/mL) by the micro-dilution method, following the recommendations of the Clinical Laboratory and Standards Institute (CLSI) 15 .The MIC was calculated and defined as the antibiotic's lowest concentration that inhibited all the visible growth of each tested isolate.After calculating the MIC, all dilutions were added to plates to count the total number of bacteria and determine the minimal bacterial concentration (MBC).MBC was defined as the lowest antibiotic concentration at which no colonies were recorded.The most resistant isolate, S. aureus S35, was selected for further studies.

Minimum Inhibitory Concentration (MIC) of MLE against S. aureus isolates
The mint stock solution was prepared in tryptic soy broth.pH was adjusted to 7.2 at 25 °C.Different concentrations of MLE extract were prepared at 800, 700, 600, 500, 400, 300, 200, 100, 50, and 25 mg/mL by diluting the stock extract in broth to achieve the desired concentrations for susceptibility testing.200 μg/mL of each dilution was added to 96-well cell culture plates.A 100 μL of the most resistant S. aureus isolate suspension was added to each well and incubated at 37 °C for 24 h.For negative controls, wells were filled with broth only with no MLE extract or antibiotic exposure.Absorbance was measured at 595 nm.As stated earlier, estimates for MIC and minimal bacterial concentration (MBC) were carried out.

Molecular identification for the most susceptible S. aureus isolates
A QIA amp DNA mini kit from QIAGEN GmbH, Hilden, Germany, was used to extract the DNA from the pure culture of the incredibly sensitive S. aureus isolate following the manufacturer's instructions.The 16S rRNA gene sequences were targeted by the universal primers 27F (5′ AGA GTT TGA TCC TGG CTC AG 3′) and 1492R (5′ TACG GCT ACC TTG TTA CGA CTT 3′).The nucleotide FASTA sequence was submitted to the NCBI GenBank under accession number (OQ766965) and NCBI database BLAST (http:// www.ncbi.nlm.nih.gov/ BLAST).Mega 11 software was used to view the distance tree for the sequences, and it was then used to build a neighbor-joining phylogenetic cladogram tree with identification based on sequence similarity 17 .

MLE's impact on the mortality of C. pipiens larvae
For the current study, 25 larvae of each size (3rd to 4th instar stage) were placed in 100 mL beakers containing various concentrations of previously prepared MLE.The MLE was initially dissolved in 10% DMSO to prepare MLE stock solution (1 mg/mL).Different testing concentrations of MLE (100%, 75%, 50%, 25%, 10%, and 0%) were then prepared by diluting the MLE stock solution in water.The effect of MLE on C. pipiens larvae's viability was determined for all concentrations using Breadcrumbs as nutrients.All larvae were incubated at 25-30 °C under a 14:10 light and dark photoperiod cycle for 72h.The total larval time (days) was calculated from the first day of treatment until the death day of the larvae.The number of dead larvae in each batch was counted every day in the morning.The negative control of each experiment (treated with DMSO-distilled water) was tested three times.The mortality (using Abbott's formula) and survival rate were determined after 24, 48, and 72 h of exposure.All the test containers were kept at room temperature with no disturbance.Correct for mortality in the control treatment using Abbott's formula (% test mortality -% control mortality/100-control mortality × 100) 18 .

Morphological and histopathological studies of C. pipiens
Based on the MLE's impact on C. pipiens mortality result, the morphological changes of the treated larvae were determined using a Labomed microscope (Labomed® Microscopes Manufacturers & Suppliers, Labo America Inc.) (at 40 and 100 ×).The dead larvae were counted on a microscopic slide and observed under the microscope 19 .For microscopic examination, all control and treated larvae were fixed with 3-5% formalin, then dried out using ethyl alcohol and cleaned with xylene.All prepared slide samples were fixed with a paraplast to take sections (7 µm).Eosin and hematoxylin stains were used for staining the sectioned larvae and control treatment 20 .The mid-guts of control and treated larvae were examined and photographed using a labomed microscope.

Total protein content
Total protein content was determined using a BCA assay kit (ThermoScientific).50 μL of protein standards and larval samples were added to tubes along with 450 μL water, 100 μL of 0.15% sodium deoxycholate, and 100 μL of 72% trichloroacetic acid to precipitate proteins.After centrifugation at 10,000 rpm for 15 min and removal of the supernatant, 50 μL of 5% SDS reagent was added to dissociate the precipitated proteins.1 mL of BCA reagent was then added, which reacts with protein peptides to form a purple-colored complex measured at 562 nm.
Absorbance was compared to a standard curve to calculate total protein levels per 25 larval samples.The assay involves precipitating proteins out of solution, and then resolubilizing them to allow colorimetric detection as a measure of total protein content 21 .

Total carbohydrates content
For quantification of total carbohydrates, the glucose standard for total soluble carbohydrates assay was carried out 21 .Briefly, a glucose stock solution of 1 mg was dissolved in 1 mL of distilled water, from which the following concentrations were prepared: 400, 200, 100, 50, 25, and 12.5 µg/mL.Larvae samples were homogenized using a sterilized mortar and then centrifuged at 10,000 rpm.Pellets were discarded, and the supernatant was collected and diluted by a ratio of 1:1 in distilled water.A 100 µL of concentrated sulfuric acid solution (75% v/v) was added to 50 µL of larvae sample in a glass vial.The vial was then filled with 200 µL of the anthrone reagent (5 mg in 100 µL of ethanol and 2.4 mL of 75% v/v sulfuric acid), and the temperature of the oven was set at 100 °C for 5 min.The mixture was heated, and allowed to cool for five minutes at room temperature, and then the analysis was performed by transferring 100 L of the sample mixture to a 96-well plate (n = 6, three independent experiments), where the resulting green color was measured at 578 nm.Data are represented as means ± SD.The results were recorded using a FluoStar Omega microplate reader 22 .

Acetylcholine esterase activity
Donepezil standard was prepared at the concentration of 5 mM to serve as a positive control.Larvae samples were prepared as mentioned before.Acetylcholinesterase enzyme was purchased from Sigma-Aldrich from the Electrophorus electrics.Cat number: 3389.Aceylthiocholine iodide substrate and the indicator 3,3′-Dithiodipropionic acid di (N-hydroxysuccinimide ester) (DTNB) were purchased from Sigma-Aldrich.Briefly, 10µL of the indicator solution (0.4 mM in buffer (1): 100 mM tris buffer pH-7.5) was transferred to a 96-well plate followed by 20µL of enzyme solution (acetylcholine esterase 0.02U/mL final concentration in buffer (2): 50 mM tris buffer PH = 7.5 containing 0.1% bovine serum albumin).Following that, 140 mL of buffer was added, then 20 mL of the sample/standard solution (1).The mixture could stand for 15 min at room temperature.The substrate (0.4 mM acetylcholine iodide buffer (1)) was then added to each well in an instant, totaling 10 L. The plate was incubated in a dark chamber for 20 min at room temperature.At the end of the incubation period, the color was measured at 412 nm.Data are represented as means ± SD.The results were recorded using a microplate reader, FluoStar Omega 21,22 .

GC-Mass chromatography
MLE was dissolved using 3 mL ethyl acetate and 1 mL was transferred to GC vial for GC/MS analysis after evaporation.The investigation of various substances of MLE that were present in modest quantities was performed using gas chromatography-mass spectrometry.The components were identified by comparing their mass spectra and retention times to those of the authentic compounds, as well as by computer matching with the NIST and WILEY libraries and by comparing the fragmentation pattern of the mass spectral data to those reported in the literature.The analysis was performed with a GC (Agilent Technologies 7890A, Poway, CA) interfaced with a mass-selective detector (MSD, Agilent 7000, Poway, CA) equipped with a polar Agilent HP-5 ms (5%-phenyl methyl poly siloxane) capillary column (30 m 0.25 mm in diameter and 0.25 m film thickness).Helium was used as the carrier gas, with a linear velocity of 1 mL/min.The injector and detector temperatures were 200 °C and 250 °C, respectively.Volume injected was 1 μL of the sample.The MS operating parameters were as follows: ionization potential 70 eV, interface temperature 250 °C, and acquisition mass range 50-800 23 .

Acetylcholine esterase and Beta-lactamase binding interactions by molecular docking
Acetylcholinesterase from C. pipiens and Beta-lactamase from S. aureus were the two proteins examined in the study, along with their associated ligands.The X-ray crystal structure of acetylcholinesterase from Drosophila melanogaster (PDB ID 1QON) and a homology model of acetylcholinesterase from C. pipiens were obtained from the RCSB Protein Data Bank and SwissModel repository respectively.These existing insect AchE structures were used as targets for docking.The proteins' 3D model structures were obtained from the UniProt KB database, improved using ModRefiner to increase the protein quality, and their active sites were predicted using Deepsite 24 .The ligands' structures were retrieved from the PubChem database, and their 3D structures were energy-minimized using Avogadro 1.2.0 software 25 .Docking simulations were performed using AutoDock Vina software with a grid box size of 20 × 20 × 20, and molecular dynamics simulations were generated using the MOE 2015 software and the SIBioLead web server.ADMET analysis was performed using the ADMETlab 2.0 web server 26 , and pathway analysis was conducted using the STITCH database.Finally, a 3D-QSAR analysis was performed using the Cloud 3D-QSAR web tool by integrating the SMILES codes for each compound with a pIC 50 value.www.nature.com/scientificreports/

Statistical analysis
All samples and collected data were statistically analyzed using IBM® SPSS® Statistics software (2017).A Tukey test was conducted with a P-value of 0.05 27 .

Ethical statement
This article does not contain any studies with human participants or animals performed by any of the authors.It was approved by the ethical committee of Faculty of Agriculture, Ain Shams University, Cairo, Egypt.

Antimicrobial potential of commercial antibiotics against clinical S. aureus isolates
In the current study, 65 S. aureus isolates were gathered from field larvae midgut samples that were collected near three hospitals in Cairo, Egypt.A susceptibility test was performed using the disc diffusion method.Of the 65 collected isolates, 18 (27.69%)were resistant to tetracycline, 20 (30.76%) were resistant to gentamycin, 12 (17.46%)were resistant to ciprofloxacin, and fifteen (23.08%) were resistant to MLE, as illustrated in Fig. 1.
As shown in Table 1, MLE's inhibitory activity was recorded as inhibition zone diameter (IZD) expressed in milliliters.Antibiotic susceptibility testing was performed against a control S. aureus strain and the selected S. aureus S35 isolate.With recorded inhibition zone diameters ranging from 2.4 to 5.2 cm, S. aureus isolate S35, one of the clinical isolates obtained from the mosquito larvae, was identified as the most resistant to the undiluted MLE out of all the isolates tested.Inhibition zone diameters (IZD) in cm were measured to assess the degree of growth inhibition.The control S. aureus strain displayed susceptibility to the antibiotics and mint extract with larger IZDs.The isolate S35 showed reduced IZD sizes, indicating decreased susceptibility and antimicrobial resistance.The results demonstrate the potential antibacterial activity of mint extract against S. aureus but also reveal challenges with antibiotic resistance strains.IZD = Inhibition zone diameter, Conc.= concentration, R = Resistant, I = intermediate, S = sensitive.All values are the mean of three replicates ± standard deviation (SD) according to Tukey's test at confidence 5 27 .

Inhibitory activity of MLE against multidrug-resistant S. aureus isolates
The results showed that undiluted MLE could inhibit a subset of the multidrug-resistant S. aureus isolates.Specifically, MLE at concentrations of 300 and 700 μg/mL inhibited 35 out of the 45 multidrug-resistant isolates tested, representing 77.77% resistance.According to data in Fig. 2, undiluted mint inhibits 61.53% of the multidrug-resistant S. aureus isolates.However, Mint could inhibit none of these isolates at dilutions ≤ 50 μg/mL.

Minimal inhibitory concentrations (MIC) for antibiotics and MLE
MIC value of the antibiotics and mint leaf extract (MLE) was determined against the S. aureus isolates using a microdilution assay, Fig. 3. S. aureus ATCC 29737 served as a positive control.The results showed isolate S35 was susciptable to the antibiotics at concentrations of 0.25-5 μg/mL.In contrast to ciprofloxacin, which had a MIC of 0.25 μg/mL for isolate S35, MLE had a higher MIC of 700 μg/mL.This demonstrated the relative efficacy of the antibiotics compared to MLE, with the antibiotics being effective at much lower concentrations than the mint extract.

Effect of MLE on the Mortality of C. pipiens larvae
The larvicidal effect of MLE on C. pipiens larvae was demonstrated in Fig. 5. Gradually raising the concentrations of the mint extract had a lethal (larvicidal) effect that, after 72 h, was rated from low to moderate (or slightly high), reaching 68%.After exposure for 72 h, 100% of MLE experienced 100% mortality, indicating a particularly strong effect at that point.After exposure for 72 h, 100% of MLE experienced 100% mortality, indicating a particularly strong effect at that point.

Morphological changes of C. pipiens larvae treated with MLE
The third and fourth instars of control larvae of C. pipiens exposed to MLE for 72 h (A) show microscopic changes in the head, thorax, midgut, and anal gill parts (B-F).The toxic effects of 20-100% MLE on C. pipiens include loss of external hairs, the epithelial layer's outer cuticle crumbling, abdominal breakage, and larval shrinkage in addition to toxic effects on various body parts (thorax, midguts, and anal gills) (Fig. 6).

Chemical determinations
The third and fourth larval instars of C. pipiens were exposed to 100% MLE for 72h at 25 °C, and the amount of total protein, total soluble carbohydrates, and acetylcholine esterase activity were measured to determine the chemical changes that occurred.As shown in Fig. 8, it was recorded that on the third day, the protein content dropped dramatically, from the calculated control treatment value of 7.963 mg/25 larvae to just 1.661 mg/25 larvae.Between 605.10 ± 11.18 µgG / 25 larvae and 341.91 ± 12.17 µgG / 25 larvae, there was a drop in the amount of total carbohydrates.Acetylcholine esterase activity dropped from 32.44 ± 4.81 U/25 larvae to 28.96 U/25 larvae.

Gas chromatography
GC-MS analysis is used to determine the production of active components in the mint leaf extract.The MLE comprises 39 chemical components as shown in Table 2.With a ratio of 35.92%, menthol was the largest active ingredient, followed by menthone (19.85%),D-Carvone (15.46%),Pulegone (5.0579%) and oxalic acid, isobutyl tetradecyl ester (4.05%).These phytochemicals have a variety of biological activities, including antibacterial, antifungal, and mutagenic potential, as well as anti-cancer properties.Moreover, they are phytotoxic and antioxidants.

Docking simulation
In contrast to carbamate in the first active site identified, the docking experiments showed that alpha guanine and cadinol had the highest binding affinity to both predicted active sites of C. pipiens acetylcholinesterase, as shown in Table 3 and Fig. 9.In active sites 2 and 1, respectively, alpha guanine displayed binding affinities of −9.3 kcal/mol and −6.8 kcal/mol followed by cadinol that showed binding affinities of −9.2 and −6.5 on active sites 1 and 2, respectively.According to these results, C. pipiens may be susceptible to alpha-guanine and cadinol's potential as acetylcholinesterase inhibitors.Alpha guanine formed alkyl and pi-alkyl interactions with the receptor in active site 1, according to the interaction analysis, whereas in active site 2, it created pi-sigma interactions and van der Waals interactions with pi-alkyl.However, cadinol formed pi-alkyl interactions on both sites.The different activities observed among the other components may be due to the presence or absence of aromaticity effects and hydrogen bonding formation.According to the "stitch" analysis, a network was generated with 12 nodes representing genes or proteins and 9 edges representing interactions between them, as shown in Fig. 10.The average node degree of 1.5 suggests that, on average, each node is connected to 1.5 other nodes in the network.The clustering coefficient of 0.925 indicates that the nodes tend to be highly interconnected with each other, forming tight clusters.The expected number of edges in the network is 12, and the PPI enrichment p-value of 0.848 indicates that the observed number of interactions in the network is not significantly different from what would be expected by chance.In other words, the network does not show evidence of being enriched for protein-protein interactions (PPIs).However, the functional enrichment analysis identified several overrepresented PFAM and INTERPRO protein domains in the network, which may suggest specific biological functions or pathways that are active in the system.

Discussion
The common house mosquito, C. pipiens L. (Culicidae), is one of the historical lymphatic filariasis vectors in Egypt and the world.The ability of Culex sp. to act as a vector for viral microbes has been extensively studied, but little is known about the bacterial and fungal microbes that live in their gut.The mosquito species C. pipiens is widespread throughout the world and is prevalent in Egypt's urban and rural areas.It is a vector that poses a risk to human public health because it spreads many pathogens, including B. cereus, B. anthrax, and S. warneri 5 , which are known to cause several illnesses, including West Nile fever, Japanese encephalitis, Dengue fever, Rift Valley fever, Bancroftian filariasis, and Avian malaria 3,13 .Despite being widely distributed, very little is known about the gut bacterial symbionts of C. pipiens 5 .Furthermore, S. aureus has been and continues to be recognized as one of the most important opportunistic pathogens in humans 7,28 .
The majority of nosocomial infections, which have been linked to continuous increases in healthcare costs, have also been caused by S. aureus antimicrobial resistance.Antibiotics are frequently administered to hospitalized patients with nosocomial infections, which promotes the colonization and infection of multidrug-resistant microorganisms 29 .It is unfortunately common for MDR pathogen infections to be associated with high rates of morbidity and mortality, making it essential to quickly identify any mutant isolates and assess their susceptibility profiles to properly direct treatment.
In this study, we evaluated the MDR ubiquity and antibiotic susceptibility patterns of the most recovered pathogens of S. aureus obtained from C. pipiens L., insect midgut specimens, around three hospitals located in Cairo governorate, Egypt.A susceptibility test for antibiotics was performed on 100 isolates.Isolates with multidrug resistance to two or more commonly used commercial antibiotics 30 .All MDR S. aureus isolates (except five) were shown to be responsive to Mint leaf at MIC ≤ 700 μg/mL in our investigation.Additionally, the data  www.nature.com/scientificreports/from this study showed that, despite several other documented cases from patients in this area, the MLE has good antibacterial activity when used in a variety of concentrations against isolated MDR S. aureus.Multiple factors, including variations in susceptibility testing procedures and extraction processes, may be responsible for these results.It states that the polysaccharides in the mint extract have medicinal properties, including immunostimulant, anti-inflammatory, wound healing, stimulation of hematopoiesis, and anti-oxidant effects.Mint contains many pharmacologically active substances, such as menthone and polygons.Anopheles culicifacies, which are responsible for 70-75% of malaria transmission in the northern rural areas of India, were found to be strongly attracted to the essential mint leaf.According to our research, the high mortality action of MLE could be attributed to its active ingredients, including menthol, Neither the full power of natural products nor their exact mechanisms of action are fully understood by us.
Scientists from all over the world are becoming more and more interested in natural antimicrobial phytochemicals, and they are working to understand these ingredients' mechanisms in depth.To provide more proof, Table 3. Binding affinity, the total number and sites of hydrogen bonds, and pi-pi stacking formed between the ligands and the protein residues at the donepezil binding domain.it is crucial to continue these studies with a sizable sample.Such work is crucial for the region's overall health as well as the local community.Mint has potent larvicidal and antibacterial properties that are effective against C. pipiens and the S. aureus that reside in their midgut.
According to the World Health Organization (WHO), 80% of people in developing countries 3 used traditional therapies.As they play a significant role as antimicrobial and anti-inflammatory agents, they rely on the use of medicinal herbal plant extracts to treat various infectious diseases 4 .
Since it was discovered that mint leaf extract has inhibitory potentials against a variety of pathogenic bacteria, including Escherichia coli, Bacillus subtilis, Salmonella typhi, Pseudomonas sp., and Klebsiella epidermidis 31 , it is the most significant antibacterial compound used by many nations in burn treatments.It was demonstrated that mint had high antibacterial activity in a study of its inhibitory activities and MIC.Furthermore, a few of the with the pi-electron density of an aromatic ring, and they are distinguished by the perpendicular orientation of the two interacting components.Van der Waals interactions are weak interactions that occur between atoms or molecules due to fluctuations in their electron density.Pi-alkyl interactions refer to the interaction between an aromatic ring and an alkyl group.These interactions suggest that alpha guanine may form a stable complex with Acetylcholinesterase in Culex pipiens through a combination of pi-sigma and van der Waals interactions.

Figure 2 .Figure 3 .
Figure 2. Inhibitory activity of mint concentrations against all S. aureus isolates collected from C. pipiens midguts.

Figure 4 .
Figure 4.The phylogenetic tree of 16S rRNA gene sequences of S. aureus MICBURN as compared to 25 strains recorded in GenBank with gene accession number OQ766965.

Figure 7 A
-F depicts the normal brush border, cell membrane, and cytoplasm of control larvae.

Figure 7 .Figure 8 .
Figure 7. Histopathology malformations of larvae treated with MLE in the third and fourth instars of C. pipiens.A: The control midgut epithelial cells' transverse section (TS); BM: basement membrane adherent to epithelial cells; N: spherical nucleus; Mv: brush border microvilli; Pm: peritrophic membrane; GL: gut lumen.Magnification strength for microscopic objects (x = 400).B-E: Transplantation staining (TS) of the midgut epithelium in larvae treated with 100% MLE showed the loss of microvilli (Mv), the peritrophic membrane (Pm), and epithelial cell death (x = 400).

Figure 9 .
Figure 9. 3D and 2D representations of the active sites, as seen using the BIOVIA discovery studio.

Figure 10 .
Figure 10.The "stitch" analysis, with generated 12 nodes representing genes or proteins and 9 edges representing interactions between them. )

Table 1 .
Antibiotic susceptibility test for MLE and the three commercial antibiotics against the S. aureus S35 isolate and the positive control strain S. aureus ATCC 29737.

Table 2 .
Spectral analysis of the most active compounds found in a mint (Mentha routundifolia) MLE sample from a GC analysis.These compounds include menthol, menthone, pulegone, and m-mentha-1,8-diene.